Project summary: Current influenza vaccine strategies, including whole or split inactivated vaccines lacking adjuvants or cold-adapted intranasal vaccination induce antibody responses that are generally short-lived and that have variable immunogenicity, particularly in young children and the elderly. The addition of adjuvants to influenza vaccine has been proposed to enhance the quality and durability of the immune response. Adjuvants are heterogeneous compounds that can enhance or alter immune responses to vaccination. This application proposes to study the immunologic mechanisms of a novel adjuvant, cationic- DNA-lipid-complexes (CLDC), in influenza vaccination. CLDC consists of a non-coding DMA plasmid combined with cationic liposomes. Specifically, we will examine the effects of this adjuvant on the enhancement of antibody responses from mouse and human B cells, both in quantity and quality. The role of Toll-like receptor (TLR) engagement in CLDC vaccination, both in dendritic cells and directly in B cells, will be studied by vaccinating mice with gene disruptions to TLR signaling (MyD88-/-Trif-/-). TLRs are pathogen recognition receptors thought to be vital in the function of many adjuvants. The role of TLR interaction with CLDC directly in B cells and dendritic cells will be examined in vivo using murine adoptive transfer experiments. In addition, the role of specific antibody isotypes and B cells in mediating influenza virus clearance and reducing immunopathology after infection will be examined through the transfer of specific anti-influenza antibody isotypes or influenza-specific B cells to nave recipients. In particular, we will examine the importance of potential immunoregulatory mechanisms of B cells in influenza infection. Finally, the ability of CLDC to activate human nave B cells and induce antibody isotype switching in vitro will be studied to determine its potential as a human vaccine adjuvant. A thorough understanding of the immunologic mechanisms of CLDC will determine its potential as an influenza adjuvant both in dose-sparing strategies or improving the quality of antibody responses. Relevance: This application aims to study Cationic-Lipid-DNA-Complexes (CLDC), a new vaccine adjuvant that can boost the immune response to influenza vaccination. An effective adjuvant for influenza vaccination could increase the effectiveness or durability of influenza vaccination. This in turn could stretch the existing vaccine supply during shortages or prolong the protective antibody response potentially decreasing the frequency of vaccination. These studies aim to determine the mechanisms by which CLDC increases the antibody response to influenza vaccination in mouse models and in human lymphocytes.